Intravital and Kidney Slice Imaging of Podocyte Membrane Dynamics

Abstract

In glomerular disease, podocyte injury results in a dramatic change in cell morphology known as foot process effacement. Remodeling of the actin cytoskeleton through the activity of small GTPases was identified as a key mechanism in effacement, with increased membrane activity and motility in vitro However, whether podocytes are stationary or actively moving cells in vivo remains debated. Using intravital and kidney slice two-photon imaging of the three-dimensional structure of mouse podocytes, we found that uninjured podocytes remained nonmotile and maintained a canopy-shaped structure over time. On expression of constitutively active Rac1, however, podocytes changed shape by retracting processes and clearly exhibited domains of increased membrane activity. Constitutive activation of Rac1 also led to podocyte detachment from the glomerular basement membrane, and we detected detached podocytes crawling on the surface of the tubular epithelium and occasionally, in contact with peritubular capillaries. Podocyte membrane activity also increased in the inflammatory environment of immune complex-mediated GN. Our results provide evidence that podocytes transition from a static to a dynamic state in vivo, shedding new light on mechanisms in foot process effacement.

Keywords: cytoskeleton; glomerular disease; podocyte.

Figure 1.

CA-Rac1 expression leads to morphologic changes in podocyte structure in vivo. Three-dimensional reconstructions of Z stacks from intravitally imaged glomeruli in (A) Confetti/Podo:Cre mice and (B) CA-Rac1/NEFTA mice after 4 days of doxycycline treatment. (C and D) Three-dimensional reconstructions of podocytes in vibratome–cut kidney slices in an organ bath from (C) Confetti/Podo:Cre mice and (D) CA-Rac1/NEFTA mice after 4 days of doxycycline treatment. In (A–D) glomerular capillaries were highlighted via a single injection of DyLight 594-Labeled Tomato Lectin. Podocytes are visible in yellow (YFP), blue (CFP), red (RFP), green (GFP) in Confetti/Podo:Cre mice or green (GFP) in CA-Rac1/NEFTA mice. Quantification of (E) podocyte perimeter and (F) area in flattened Z stacks from Confetti/Podo:Cre and CA-Rac1/NEFTA mice (n=36 podocytes versus n=37 podocytes, respectively, from at least three individual animals imaged intravitally or in vibratome-cut kidney slices). ***P≤0.001.

Figure 2.

Podocytes are stable cells in vivo, and Rac1 overactivity increases podocyte membrane dynamics. Shown in each row are time course images representing three-dimensional reconstructions of Z stacks acquired at 0, 3, and 6 minutes; total imaging periods were up to 49.5 minutes. (A) Intravitally imaged glomeruli in Confetti/Podo:Cre mice show stable podocyte processes. Membrane movement was visualized by using a heat map (column 4) depicting the pixel intensity change in a flattened Z stack over 15 minutes. Because the two cells depicted expressed different fluorophores, the analysis for each channel was done separately, and the pictures were combined (dashed line). (B) Under the same conditions, CA-Rac1-expressing podocytes showed increased membrane ruffling, indicated by a higher intensity in the heat map along the cell borders. (C and D) Imaging of kidney slices in an incubation chamber allowed for more detailed visualization of the stable three–dimensional structure of major processes in (C) Confetti/Podo:Cre podocytes, whereas (D) CA-Rac1/NEFTA podocytes showed rapid rearrangement of short, lamellipodia–like protrusions. Please note that changes in the focal plane caused by heartbeat or tissue drift can create small artifactual movements that are detected as a background pixel intensity change as seen in the heat maps in A and C.

Figure 3.

Podocytes expressing CA-Rac1 are shed and can be detected within tubules. (A) Intravital imaging of a glomerulus in a CA-Rac1/NEFTA mouse after 4 days of doxycycline treatment reveals an GFP-positive podocyte (green) within a tubule (arrow). (B) Intravital imaging of a CA-Rac1–expressing podocyte actively migrating within a tubule and extending lamellipodia (arrows). (C) Intravital three–dimensional reconstruction of a podocyte that has integrated into the tubular epithelium. Whereas much of the cell body remained in the tubular lumen (T), the podocyte (P; green) contacted the DyLight 594-Labeled Tomato Lectin intertubular blood vessels (V; red), which is highlighted by arrows.

Figure 4.

Increased membrane dynamics are a feature of injured podocytes in an inflammatory environment. (A) Time course of three-dimensional reconstructions of Z stacks in a vibratome–cut kidney slice from a healthy Confetti/Podo:Cre mouse. (B) Time course of three-dimensional reconstructions of Z stacks in a vibratome–cut kidney slice from a Confetti/Podo:Cre littermate at day 3 after injection of nephrotoxic serum. Note the membrane movement in the RFP-expressing cell (orange; circle). (C) Quantification of pixel intensity changes over 15 minutes in vibratome–cut kidney slices. Pixel intensity changes in representative glomerular areas of 25×25 μm (12 representative areas from at least three individual animals per group) were quantified. Shown are the average maximum intensity changes on the y axis and the percentage of pixels on the x axis. A sigmoidal curve was fitted to the data, and half-maximum percentages were determined. For analysis of statistical significance, values on the x and y axes at the half-maximum from each area were compared separately between the three groups. CA-Rac1/NEFTA and nephrotoxic serum–injected Confetti/Podo:Cre mice showed significantly increased movement versus controls. **P<0.01; ***P<0.001.